PROJECT SUMMARY/ABSTRACT The goal of this project is to decipher the mucin glycan code that regulates microbial virulence. A layer of thick, well-hydrated mucus is a key defense mechanism on epithelial linings such as on the mouth, gastrointestinal tract, and lungs. The exceptional molecular diversity and complexity of glycans associated with mucin polymers, the gel-forming building blocks of mucus, has been recognized for decades. However, their potential for regulating interactions between a host and its associated microbes has barely been tapped because the individual bioactivities of glycans have been intractable to analysis. Our results from the past funding period support a central role for mucin glycans in host protection by regulating cross-kingdom virulence; our results also strongly support the relevance and feasibility of the proposed efforts to identify the glycan structures and mechanisms responsible for antivirulence effects. We propose to combine functional analysis with bottom-up engineering of mucin-like glycans and polymers to unravel the design principles of glycan signals and the mucin regulatory code. This knowledge will empower us to begin to elucidate, and ultimately harness, the myriad biological consequences of glycans and mucins on microbes and their hosts. In Aim 1, we will harvest bioactive glycans from mucins to generate annotated libraries from isolated mucin O-glycans for functional analysis from the major mucosal surfaces in the body, including the mouth, lungs, and digestive tract. These libraries will allow, for the first time, functional studies to obtain insight into mechanisms and chemistries of O- glycans that affect host-microbe interactions, such as glycan size, specific residue sequence, glycan linkages, and geometry. In Aim 2, we will identify the mechanisms by which mucin O-glycans attenuate virulence in two important human mucosal pathogens, Pseudomonas aeruginosa and Candida albicans, which are becoming increasingly resistant to treatment. In Aim 3, we will characterize the anti-virulence effects of mucin O-glycans in a well-established pre-clinical in vivo infection model. In Aim 4, we will integrate the knowledge from Aims 1- 3 to engineer prioritized O-linked glycosylated mucin-like polymers with previously unattainable precision. This project directly addresses an urgent health care problem: antimicrobial resistance is spreading rapidly and demands new approaches to combat problematic pathogens. We expect to deliver new concrete chemical design parameters and molecules to manage two problematic pathogens that are becoming increasingly resistant to treatments. The multidisciplinary team has the expertise necessary for combining fundamental science questions with pre-clinical validation and cutting-edge engineering applications: a biologist with experimental and theoretical expertise in mucus hydrogel systems, a microbiologist with expertise in in vivo infection models, and a chemist with expertise in controlled glycan and polymer synthesis and characterization.